Epidemiological, genomic and evolutionary characteristics of G9P[8] group A rotavirus in Chinese diarrheic children from 2018-2020

Epidemiological, genomic and evolutionary characteristics of G9P[8] group A rotavirus in Chinese diarrheic children from 2018-2020 | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 July 2025 V1 Latest version Share on Epidemiological, genomic and evolutionary characteristics of G9P[8] group A rotavirus in Chinese diarrheic children from 2018-2020 Authors : Rui Peng , Mengxuan Wang , Jinbo Xiao , Ranran Cao 0000-0001-5809-4714 , Caixia Li , Xiang Li , Xiaozhou Kuang , … Show All … , Yihui Cao , Meirong Tang , Zhongyan Fu , Yugeng Zhang , Xiao Hu , Wenna Zhao , Peng Wang , Dandi Li [email protected] , and Zhaojun Duan Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.175242251.14507317/v1 205 views 116 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract This study investigated the epidemiological, genomic, and evolutionary characteristics of group A rotavirus (RVA) in Chinese diarrheic children from 2018 to 2020 and infer the vaccine efficacy. Using data from China CDC’s National Viral Diarrhoea Surveillance Program , 14,217 stool samples from children <5 years with acute gastroenteritis were tested by ELISA and G/P genotyping, revealing a 23.62% RVA positivity rate. G9P[8] dominated (69.51%), with autumn and winter seasonal peaks. Thirty-eight RVA-positive samples underwent RT-PCR-based whole-genome sequencing, identifying two major genotype constellations (G9P[8]-E1/E2) by RotaC_v2.0. Identity and phylogenetic analysis with MEGA11 and DNAStar v.11.1 showed that VP7/VP4 lineage had significant genetic divergence from vaccine strains. The evolutionary rates of VP7 and VP4 were estimated to be 1.402 × 10 -3 (VP7) and 6.924 × 10 -4 (VP4) substitutions/site/year by TempEst v.1.5.3 and BEAST v.1.10.4 software. Neutralizing epitope analysis revealed amino acid variations between Chinese G9P[8] circulating strains and vaccines through BioEdit v.7.0.9.0 and PyMOL v.3.1.3, potentially impacting vaccine immunogenicity. Epidemiological, genomic and evolutionary characteristics of G9P[8] group A rotavirus in Chinese diarrheic children from 2018-2020 Rui Peng 1 , Mengxuan Wang 1 , Jinbo Xiao 2 , Ranran Cao 3 , Caixia Li 3 , Xiang Li 3 , Xiaozhou Kuang 3 , Yihui Cao 3 , Meirong Tang 3 , Zhongyan Fu 3 , Yugeng Zhang 3 , Xiao Hu 3 , Wenna Zhao 3 , Peng Wang 3 , Dandi Li 1* , Zhaojun Duan 1* 1 National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID), NHC Key Laboratory for Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 2 Department of Poliomyelitis, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 3 Viral Diarrhea Surveillance Network, China. * Correspondence: Dandi Li ( [email protected] ) Zhaojun Duan ( [email protected] ) Full list of author information is available at the end of the article. ABSTRACK: This study investigated the epidemiological, genomic, and evolutionary characteristics of group A rotavirus (RVA) in Chinese diarrheic children from 2018 to 2020 and infer the vaccine efficacy. Using data from China CDC’s National Viral Diarrhoea Surveillance Program , 14,217 stool samples from children <5 years with acute gastroenteritis were tested by ELISA and G/P genotyping, revealing a 23.62% RVA positivity rate. G9P[8] dominated (69.51%), with autumn and winter seasonal peaks. Thirty-eight RVA-positive samples underwent RT-PCR-based whole-genome sequencing, identifying two major genotype constellations (G9P[8]-E1/E2) by RotaC_v2.0. Identity and phylogenetic analysis with MEGA11 and DNAStar v.11.1 showed that VP7/VP4 lineage had significant genetic divergence from vaccine strains. The evolutionary rates of VP7 and VP4 were estimated to be 1.402 × 10 ⁻³ (VP7) and 6.924 × 10⁻⁴ (VP4) substitutions/site/year by TempEst v.1.5.3 and BEAST v.1.10.4 software. Neutralizing epitope analysis revealed amino acid variations between Chinese G9P[8] circulating strains and vaccines through BioEdit v.7.0.9.0 and PyMOL v.3.1.3, potentially impacting vaccine immunogenicity. Keywords: rotavirus, G9P[8], whole genome, epidemiology, evolution 1 Background Rotavirus (RV) is a leading cause of diarrhoea in children under 5 worldwide. Its high incidence and death toll pose significant health threats, with ~128,500 global deaths in 2016 1–3 . Therefore, monitoring and prevention are critical. At present, 14 out of 42 RV G serotypes (G genotypes) were distributed mostly worldwide, of which G1-G4, G9, and G12 are the most common. Whereas 14 out of 54 P serotypes (P genotypes) are widely distributed, of which P[4], P[6], and P[8] are the most common (https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg). Six genotype combinations G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8] are the most common, responsible for 90% of RV infection in the world 3 . G9 emerged as a globally important strain only in the 1990s 4–6 . The prevalent rotavirus genotypes are constantly changing globally 7,8 . For example, the G9P [8] RV in France increased from 32.1% during the 2014-2015 seasons to 64.1% and 77.3% during the 2015-2016 and 2016-2017 seasons respectively, replacing G1P [8] as the predominant circulating genotype for the first time 9 . In a study in India, G9 (40%) and G2 (40.3%) replaced G1 as the most common genotypes in hospitalized cases and outpatients, respectively 10 . Currently, vaccine is the most effective measure to prevent and control rotavirus diarrhoea. There are two major RV vaccines currently in use worldwide, Rotarix, an orally attenuated live vaccine, and RotaTeq, a recombinant vaccine 11 . Although protection and safety of both vaccines were widely reported from developed countries and Latin America, their protection was relatively poor in developing countries 3 . There are two vaccines on China’s market, RotaTeq (G1, G2, G3, G4, P[8]) and LLR (G10P[15]). RotaTeq is less effective in China than in developed countries 12 . The protection rate of LLR against severe rotavirus gastroenteritis (RVGE) and all other diarrhoea levels are low (52% - 88%, against RVGE; 35%, against other levels) 13 . The effectiveness of vaccines is influenced by a variety of factors, and better access to health, nutrition, and clean water are the obvious reasons for the discrepancy between HIC and LIC 13 . However, there are other subtle reasons for the different effectiveness. Individuals are less susceptible to RV infection if they do not over-express the HBGAs, since the antigens are important targets for virus attachment to cells 14,15 . The gut microbiome is another possible reason. For example, a study on Ghania children showed higher abundance of Proteobacteria and Eggerthella with positive vaccine response and higher abundance of Fusobacteria and Enterobacteriaceae with poor vaccine response 16 . Although RV vaccines show some heterologous protection 17 , the characteristics of circulating strains, such as genotype and neutralizing antigenic properties, are other potential factors contributing to the different vaccine effectiveness. For example, the combination of prevalent G/P genotypes may be able to explain the differences in rotavirus vaccine effectiveness between high-income and low-income countries. Regions of the world primarily feature the genotype G1P[8], G2P[4], G3 P[8], G4P[8], and G9P[8], which effectively neutralised by immune response cause by Rotarix (G1P[8]) and RotaTeq (G1, G2, G3, G4 and P[8]). However, G12 and P[6] are commonly found in sub-Saharan Africa, None of which are not included in Rotarix and RotaTeq 4–6 . The difference in neutralizing antigenic epitopes may be another factor affecting vaccine efficacy. Studies have shown that the introduction of human rotavirus vaccines may alter the mechanisms and balance that drive rotavirus evolution, influencing the spread of new strains with antigenic characteristics different from those in vaccine formulations 18,19 . Similarly, in the Omicron variant, convergent evolution has frequently occurred, involving spike protein sites that are crucial for immune evasion, receptor binding, or proteolytic cleavage 18,19 . To deeply investigate the epidemiological, genomic, and evolutionary characteristics of human rotaviruses circulating in China, especially to evaluate the protective efficacy of current vaccines based on the above-mentioned characteristics, Chinese children’s diarrheic samples from 2018 to 2020 were collected and systematically analyzed. To obtain full sequences for genomic analysis, 38 stool samples were randomly selected and sequenced. This study provides insights into rotavirus epidemiology, evolution, as well as prevention and control. 2 Materials and methods 2.1 Epidemiological Data Sources, Sampling, and Rotavirus Confirmation China CDC’s National Viral Diarrhoea Surveillance Program (2007 Revision) established 26 provincial-level surveillance sites across China to monitor enteric viral infections in <5-year-olds. Between 2018-2020, 14,217 clinical samples were tested for RVA. Specifically, samples were first processed into 10%-20% faecal suspensions with HBSS, vortexed 3 min, centrifuged at 5000rpm for 10 min, and stored at -70°C. Then, the presence of RVA and the combination of G/P genotypes were detected by ELISA and G/P genotyping per China CDC’s national protocol (Supplementary Information 1). 2.2 Statistical Analysis The initial database was constructed using EpiData 3.0.6.205 software. Further processing and analysis of all information were carried out through GraphPad software. The chi-squared test is a non-parametric statistical method suitable for categorical data that can test the difference between observed and expected frequencies. In this study, the SPSS v.26.0 software was used to identify the significance of the differences in RVA detection rates (or detection rate of specific genotype, or composition ratio of specific genotype) across different months using the chi-squared test. The statistical significance was defined as p-values (≤0.05). The significance level was set at 0.05. Related calculation equations are as follows. RVA prevalence = (ELISA positives ÷ total specimens) × 100%. Genotype prevalence = (genotype count / total specimens) × 100%. Composition ratio of specific genotype= (genotype count / ELISA positives) × 100%. 2.3 RVA Fragments Amplification and Cloning for Whole Genome Sequencing In this study, China CDC archived surveillance samples collected between 2018 and 2020 were randomly selected for study. These samples came from sentinel hospitals in 11 provinces and Beijing of China CDC’s rotavirus monitoring network (Beijing, Jilin, Inner Mongolia, Hebei, Henan, Anhui, Shanghai, Jiangsu, Guangdong, Guangxi, Sichuan, and Yunnan) (Figure 1). To obtain complete sequences for genomic analysis, 40 randomly selected stool samples were sequenced, of which 38, having sufficient viral titers for whole-genome sequencing, were included in subsequent analyses (Supplementary Information 2). According to the previously described methodology 20,21 , RVAs in samples were amplified by reverse transcription polymerase chain reaction (RT-PCR), and DNA fragments were inserted into T-cloning vectors. Following sequencing analysis by Beijing TsingKe Biotech Co., Ltd., the complete rotavirus genome (11 RNA fragments) was successfully obtained. 2.4 Genotyping, Sequence Identity and Phylogenetic Analysis of RVA Fragments Sequence splicing was performed on the sequenced data of the 38 China RVA strains using SeqMan workflow in the DNAStar Bioinformatic Software Package (v.11.1). This yielded the complete eleven fragments of each RVA strain. After that, genotyping was performed on all the fragments using the web-based classification system RotaC_v2.0 (https://www.rivm.nl/mpf/typingtool/rotavirusa/). The phylogenetic and sequence identity characteristics of 38 RVA were analysed from complete genomes. Besides these, vaccines strains, pre-2020 Chinese RVA sequences, and global RVA strains were included. First, nucleic and amino acid sequence identities analyses were performed using software BioEdit v.7.0.9.0. Next, phylogeny of 11 RVA fragments were separately analysed. Before that, nucleotide-level genetic distances with best-fit models were calculated. Then, the phylogenetic reconstructions were performed by MEGA11 employing maximum-likelihood and1000 bootstraps to balance the accuracy of the phylogenetic tree and the verification of its reliability. The lineages were identified according to earlier studies for VP7 22 , VP4 23 , NSP4 24 and other gene trees 20 . 2.5 Bayesian Evolutionary Analysis of VP7 and VP4 Genes Using BESTE v.1.10.4 software, conduct in-depth analysis of the dynamic evolution characteristics of VP7 in G9 genotype and VP4 in P [8] genotype. Here, 38 rotavirus strains from China between 2018 and 2020 are used as the main analysis objects, while RVA sequences and vaccine sequences from around the world are used as reference sequences. Among them, there are 318 reference sequences for VP7 and 165 reference sequences for VP4. TempEst v.1.5.3 25 . were used for root to tip analysis to ensure data reliability. The evolution speed of genes and the characteristics of the most recent common ancestor (TMRCA) were analysed. Using Tracer v.1.7.2 software (http://tree.bio.ed.ac.uk/software/tracer) and by running MCMC 100000000 times, the effective sample size (ESS) had been increased to over 200. Next, the MCC tree was annotated and displayed in TreeAnnotator v1.10.4 and FigTree v.1.4.3, respectively (http://tree.bio.ed.ac.uk/software/figtree) 26 . The information about these sequences for VP7 and VP4 Bayesian Evolutionary analysis was listed in Supplementary Information 3 and 4. 2.6 Analysis of Neutralizing Antigenic Epitopes on Deduced VP7 and VP4 Amino Acid Sequences The corresponding antigenic epitopes of VP7 and VP4 genes obtained from diarrhoea specimens in this study were compared and analysed with epidemic RVA strains all of the world and the Group A rotavirus vaccines with the same genotype through BioEdit v.7.0.9.0. The differences in neutralizing antigenic epitopes between the specimens and vaccines were analyzed in terms of three-dimensional structural variations using protein database files 1KQR 27 and 3FMG 28 by PyMOL v.3.1.3. 3 Results 3.1 Annual and monthly prevalence of China’s RVA From 2018 to 2020, a total of 3,358 out of 14,217 paediatric stool specimens from Chinese children admitted for acute gastroenteritis under five years old were RVA-positive by ELISA, yielding a positivity rate of 23.62%. By years, the RVA detection rates among hospitalized children with diarrhoea was 27.01% (2018), 26.40% (2019), and 17.13% (2020) (Table 1). The five major globally circulating G/P combinations are G9P[8], G3P[8], G1P[8], G4P[8], and G2P[4]. Only G4P[8] was not detected in China. The G/P genotype combination predominantly in China was G9P[8] accounting for 69.51% of all RVA. Other nonglobal major genotypes make up between 0.5% (2018) to 7.02% (2020) (Table 2). Thus, in general between 2018 and 2020, there are three to four local genotypes circulating in China on top of the four global genotypes. The prevalence of local genotypes however is low. When focusing on G9P[8], the largest circulating genotype in China, the detection rates were 20.57% (2018), 17.54% (2019) and 10.60% (2020). This yield annual G9P[8] genotype composition ratio within all RVA cases between 61.90% to 76.15% (Table 3). Table 1 RVA Positive Detection Rate in China from 2018 to 2020 2018 5,294 1430 27.01% 2019 4310 1138 26.40% 2020 4,613 790 17.13% Total 14,217 3,358 23.62% Table 2 G/P Genotype Combinations of Rotavirus from 2018 to 2020 in China composition ratio (%) 69.51% 3.82% 2.46% 2.21% None 19.9% Other Genotypes 2018 2019 2020 G9P[4] 0.33% 0.61% 0 G2P[8] 0.08% 0.48% 0.76% G1P[4] 0.08% 0 0 G4P[4] 0 0.12% 0 Table 3 G9P [8] RVA infections from 2018-2020 2018 1089 20.57 76.15 2019 756 17.54 66.43 2020 489 10.60 61.90 Total 2334 16.42 69.51 The monthly distribution of diarrhoea cases caused by acute gastroenteritis in children under 5 years old showed a biphasic pattern. The epidemic curve of the cases exhibits a bimodal fluctuation pattern, forming a typical bimodal distribution in the summer half year (May-Jul) and winter half year (Nov-Jan) (Figure 2A). The prevalence of detected RVA in diarrhoea stool samples varied from 6.03% to 48.04% monthly. Notably, RVA cases exhibited a single-cycle pattern, peaking in autumn and winter (Nov-Jan) and reaching their lowest levels between summer and autumn (Jun-Sep). The monthly variation in RVA detection rates was statistically significant (Chi-Square χ² = 1448, P < 0.001). The detection of G9P[8] genotype from all acute gastroenteritis cases ranged from 1.76% to 32.63% and differed each month (Chi-Square χ² =1179, P < 0.001). Detection rates fluctuated showing a single cycle trend as was the RVA cases in general (Figure 2B). However, when aggregated specifically by RVA cases, the G9P[8] genotype didn’t show clear seasonal cycle and instead peaked erratically in January, May, July, and October (Figure 2C). The differences among G9P[8] composition ratios by month were statistically significant (Chi-Square χ² =172.4, P<0.001). 3.2 Genotyping All amplified sequences have been submitted to GenBank. Results showed that 24 samples belonged to the most common Wa-like genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 (abbreviated as G9P[8]-E1), account for 63.2% of all samples. Ten belonged to G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (abbreviated as G9P[8]-E2), accounting for 36.8% (Supplementary Information 5). 3.3 Identity and phylogenetic trees Identity and phylogenetic analysis of VP7, VP4, VP6, and NSP4 Identity and phylogenetic analyses were carried on VP7, VP4, VP6, and NSP4 of the 38 Chinese RVA strains from 2018 to 2020 (Supplementary Information 6, Figure 3). The analyses revealed varying levels of conservation among the 38 strains. For nucleotide sequences, VP6 showed the highest identity at 96.7%-100%, followed by VP7 at 92.0%-100% and VP4 at 88.4%-100%. In contrast, NSP4 exhibited the greatest variation, with nucleotide sequence identity ranging from 81.6% to 100%. For amino acid sequences, VP6 again demonstrated the highest similarity at 99.2%-100%, followed by VP7 at 95.3%-100% and VP4 at 92.6%-100%. NSP4 showed the lowest similarity, with values ranging from 82.8% to 100%. The findings highlight sequence variability in the VP4 and NSP4 fragments of the 38 strains compared to VP7 and VP6 (Supplementary Information 6). In the phylogenetic tree, G9 genotype RVA VP7 genes were divided into six lineages (Figure 3A) 28 . Global G9-VP7 genes today are mainly in lineages III and VI; others have older strains and vaccines. In this study, 33 RVA strains were in lineage VI, like most recent Chinese RVA strains. They’re closest to 2018 - 2019 G9P[8]-E2/E1 epidemic strains in China and Japan (e.g., Z2768, E6398, L2448, Tokyo18-30, Tokyo18-43). The other 5 strains were in lineage III, forming a branch with other 2018 - 2019 Chinese G9P[8] strains (e.g., Fuzhou18–152 and Fuzhou19-84). All 38 strains are distantly related to G9 vaccine strains ROTAVAC - 116E (lineage II) and ROTASIIL-Au32 (lineage I), with 74.6%/74.8% nucleotide and 78.2%/92.6% amino acid sequence identities respectively. For VP4 gene phylogenetic analysis, P[8] genotypes were divided into four lineages (Figure 3B) 29 . Lineages 1 and 2 had mainly old and vaccine strains, while most modern P[8] (e.g., G3P[8], G9P[8], G1P[8]) were in lineage 3, with few in lineage 4. The 37 strains clustered in lineage 3, close to 2017-2019 G9P[8]-E2/E1 epidemic strains in China and Japan (97.5–99.9% nt, 98.7–100.0% aa identities), such as Tokyo18-30, Fuzhou18-152, and Tokyo16-4754. One strain (SD18370121) in lineage 4 was related to Pakistan’s G9P[8] strain HF66 (90.9% nt, 92.0% aa identities). All 38 study strains were distant from P[8] vaccines (RotaTeq and Rotavin-M1 in lineage 2, Rotarix in lineage 1), sharing 89.2–93.2% nt and 92.0–96.6% aa identity with them. In the VP6 phylogenetic tree, I[1] VP6 genes were divided into four lineages (Figure 3C). Lineages 2-4 mainly had old and vaccine strains with G3P[8], G1P[8], G9P[8], and G12P[8] genotypes. Most modern I1 RVA strains (G1P[8], G3P[8] etc.) clustered in I1 - lineage 1, sharing 90.0-100.0% nt and 93.4-100.0% aa identities. All 38 RVA strains were of I1 genotype. 32 clustered in Clade A, which had 2018-2019 Chinese/Japanese G9P[8]-E2 strains (e.g., E6398, L2448, Tokyo18-30, Tokyo18-38, Tokyo18-27), with 98.2-100.0% nt and 99.2-100.0% aa identities. The other 6 strains were in Clade B-1, sharing 99.6-100.0% nt and 100.0% aa identities. They were close to China’s 2019 G9P[8]-E2 strain E6356 and conventional I1 strains on Clade B (e.g., L1621, G3P[8]). In the NSP4 phylogenetic tree (Figure 3D), 25 of 38 RVA strains were of E1 genotype, and 13 were of E2 genotype. E1 had three lineages (I, II, III), with contemporary E1 strains in all. E1-Lineage I had old and vaccine strains (mostly G1P[8]), E1-Lineage II had G3P[8] strains, and E1-Lineage III had G9P[8] and G3P[8] strains. Chinese E1 strains were in Lineage II and III, and the 25 E1 strains in this study were all in Lineage III, closest to Chinese strains from 2012-2013 (82.1%-99.8% nt, 84.0%-100% aa identities). E2 had four lineages. Lineages I and III were of old strains and vaccines. Lineage II’s E2 strain was of middle ”age”. Recent E2 strains were mainly in E2-Lineage IV, with two sub-branches (Clade A and B). Clade A had 2018-2019 Chinese/Japanese G9P[8]-E2 strains and Japanese G2P[4]-E2 strains. Clade B had G9P[8]-E2 strains from different regions. All 13 Chinese 2018-2019 G9P[8]-E2 strains were in Lineage IV-Clade A, closest to 2018-2019 Chinese/Japanese G9P[8]-E2 and G2P[4] strains (81.2–100.0% nt, 82.8–100.0% aa). China had few G2P[4] RVs. G1P[8], G3P[8], G2P[4] RVs from multiple countries made up Lineage IV-Clade B, including Japanese reassorted G3P[8]-E2 strains from 2015-2018. Identity and phylogenetic analysis of other structural protein coding genes Identity and phylogenetic analyses were carried on VP1-VP3 of the 38 Chinese RVA strains from 2018 to 2020 (Supplementary Information 7, Figure 4A-C). For VP1 (Figure 4A), all 38 strains were of R1 genotype, clustered on one branch, with 97.8–100.0% nt and 98.6–100.0% aa identities. They were close to 2018-2019 Chinese and Japanese G9P[8]-E1 and new G9P[8]-E2 strains (e.g., L2448). For VP2 (Figure 4B), the 38 strains were of C1 genotype, sharing 98.1–100.0% nt and 99.3–100.0% aa identities. They were related to 2016-2019 Chinese and Japanese G9P[8]-E1 and new G9P[8]-E2 strains (e.g., Z2768). For VP3 (Figure 4C), all 38 strains were of M1 genotype, with 98.1–100.0% nt and 99.3–100.0% aa identities. They were closest to 2018-2019 Chinese and Japanese G9P[8]-E2 strains (e.g., E6398). Identity and phylogenetic analysis of other non-structural protein coding genes Identity and phylogenetic analyses were carried on NSP1-NSP3 and NSP5 of 38 Chinese RVA strains from 2018 to 2020 (Supplementary Information 7, Figure 4D-G). For NSP1 (Figure 4D), all 38 strains were of A1 genotype, with 83.5–100.0% nt and 81.2-100.0% aa identities. 37 strains were close to Japanese/Chinese G9P[8]-E1/E2 strains from different years, and 1 was close to Chinese strains in 2017-2018. For NSP2 (Figure 4E), the 38 strains were of N1 genotype, sharing 98.3–100.0% nt and 98.1–100.0% aa identities. They were closest to G9P[8]-E2/E1 strains in China, Japan, and some G3P8 and G1P[8] strains. For NSP3 (Figure 4F), all 38 strains were of T1 genotype, with 93.8–100.0% nt and 97.0–100.0% aa identities. 35 strains clustered on Clade A, close to Chinese/Japanese G9P[8] strains, and 3 on Clade B, close to strains from other countries. For NSP5 (Figure 4G), all 38 strains were of H1 genotype, sharing 97.4–100.0% nt and 97.9–100.0% aa identities, mainly close to Chinese/Japanese G9P[8] strains. 3.4 Bayesian evolutionary analysis of VP7, VP4 Temporal signal analysis conducted with TempEst v.1.5.3 demonstrated both inverse and direct relationships between genetic distance and temporal distribution (branch length of phylogenetic tree) for VP7 and VP4 sequences (Figure 5A and Figure 5B). It suggested the dataset had a temporal signal. However, the low R 2 values indicated poor fit with the molecular clock. This was explainable by sampling issue used to establish phylogenetic tree in this study. Majority of the sequences came from China between 1976 and 2020. This was intentional because the interest of the study was China’s sequences. Thus, the subsequent evolutionary rate estimation framework was advanced using Bayesian inference employing tip-calibrated molecular clocks. The phylodynamic of G9P[8] RVA was characterized. The VP7 gene of G9 genotype rotaviruses exhibited a mean nucleotide substitution rate of 1.402 × 10 -3 (95% HPD: 1.1023 × 10 -3 - 1.7053.35 × 10 -3 ) substitutions per site per annum. The time of the most recent common ancestor (TMRCA) for G9 strains used in this study was around year 1921 (TMRCA = 1920.61; 95% HPD interval: 1867.63-1954.17). The VP4 gene of P[8] genotype rotaviruses exhibited a mean nucleotide substitution rate of 6.924 × 10 -4 (95% HPD: 5.74 × 10 -4 to 8.13 × 10 -4 ) substitutions per site per annum. The TMRCA for the 165 P[8] strains was estimated to emerge in 1826 (TMRCA = 1825.86; 95% HPD interval: 1732.05-1904.08). The MCC trees of VP7 and VP4 were generated using the Markov Chain Monte Carlo (MCMC) approach (Figure 5C and Figure 5D). Evolutionary reconstruction revealed two distinct clustering patterns: Chinese G9P[8] RVAs partitioned into two dominant VP7 lineages (III and VI), emerging circa 1981, with an ancestral clade (Clade C) splitting earlier in 1973 (Figure 4C). Conversely, VP4 phylogeny showed segregation into two sub-lineages within lineage III, originating approximately 2005 (Figure 4D). It is noteworthy that strain SD18370121 was detected in lineage IV, which is rarely observed in Chinese RVA strains (Figure 4D). 3.5 Analysis of deduced neutralizing antigenic epitopes Profiling of predicted neutralizing epitopes showed distinct VP7/VP4 epitope structures in 38 Chinese G9P[8] RVA strains versus global G9 variants and vaccines (Tables 4 and Table 5). VP7 of rotavirus has 29 neutralizing epitopes in three regions (7-1a, 7-1b, and 7-2) 30 . Among 38 Chinese strains, only two amino-acid differences at sites 100 and 221 were seen. In Chinese G9P[8] isolates, three substitutions at positions 960, 100, and 221 were identified. VP7 phylogenetic analysis placed 33 Chinese strains in lineage VI and 5 in lineage III. Epitope analysis revealed minimal amino-acid variations (1 or 2) between Chinese and circulating strains within each lineage. The G9 ROTASIIL vaccine differed from the 33 Chinese lineage-VI RVA strains by three amino acids (87, 100, 242) in the 7-1(a and b) region. Substitutions at 87 and 100 aa might influence the vaccine’s immunogenicity. Between ROTASIIL and 5 lineage-III strains, there were two amino acid variations (87, 242). The A87T change could impact vaccine’s immune response. The G9 vaccine ROTAVAC differed from 33 Chinese lineage-VI RVA strains by three amino acids (87, 100, 145) in 7-1a and 7-2 regions, showing characteristics of immune evasion variants. Between ROTAVAC and 5 lineage-III strains, there were three amino acid variations too (87, 100, 145), exhibiting characteristics of escape mutant. Comparing deduced VP7 epitopes of 38 Chinese RVAs to those of commercial vaccines of various G genotypes (RV1-G1, RV5-G1/G2/G3/G4, ROTAVIN-M1-G1, LLR-G10) showed 12–16 amino acid differences in 33 lineage-VI strains and 11–18 differences in 5 lineage-III strains. Table 4 Predicted VP7 neutralizing antigen epitope polymorphisms of 38 RVA isolates in China compared to vaccines and global G9 circulating reference isolates. 87 91 94 96 97 98 99 100 104 123 125 129 130 291 201 211 212 213 238 242 143 145 146 147 148 190 217 221 264 33 Chinese RVAs-Lineage VI T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G E6398-CHN-G9-E2-2019 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G Fuzhou19-58-CHN-G9-E1-2019 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G JZ1810-CHN-G9-2018 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G Km15119-CHN-G9-2016 1 T T G T E W K N Q D T I D K Q N T A D N K D S T L S E S G Km15100-CHN-G9-2015 1 T T G T E W K N Q D T I D K Q N T A D N K D S T L S E S G SC8-CHN-G9-2014 1 T T G A E W K N Q D A I D K Q N T A D N K D S T L S E S G SC6-CHN-G9-2013 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G BJ-Q794-CHN-G9-2012 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G BJ-Q532-CHN-G9-2011 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G Tokyo18-43-JPN-G9-E2-2018 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G NS17-A959-RUS-G9-2017 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G 3000053718-USA-G9-2015 1 T T G T E W K N Q D T I D K Q N T A D N K D S T L S E S G 3000014466-USA-G9-2014 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G 1CR24-THA-G9-2015 1 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G 5 Chinese RVAs-Lineage III T T G T E W K D Q D A I D K Q N T A D N K D S T L S E G G Fuzhou19-84-CHN-G9-E1-2019 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E G G Fuzhou18-152-CHN-G9-E2-2018 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E G G BJ-Q33-CHN-G9-2010 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E G G LL09131481-CHN-G9-E1-2009 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E S G L169-CHN-G9-2000-2006 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E S G CAU10-55-KOR-G9-2010 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E G G SP014-JPN-G9-2013 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E S G 3468-KEN-G9-2016 2 T T G T E W K D Q D A I D K Q N T A D N K D S T L S E S G 3000558285-USA-P8-2016 2 A T G T E W K D Q D A I D K Q N T A D N K D S T L S E S G JES11-ITA-G9-2010 2 T T G T E W K N Q D A I D K Q N T A D N K D S T L S E S G Rotasiil-AU32-G9 3 A T G T E W K D Q D A I D K Q N T A D T K D S T L S E S G Rotavac-116E-G9 3 I T G T E W K G Q D A I D K Q N T A D N K N S T L S E N G RV1-G1 4 T T N G E W K D Q S V V D K Q N V D N T K D Q N L S M N G Rotavin-M1-G1 4 T T S G E W K D Q N V V D R Q N V D N T K D Q N L S T N G RV5-G1 4 T T N G D W K D Q S V V D K Q N V D N T K D Q S L S M N G RV5-G2 4 A N S D E W E N Q D T M N K Q D V S N S R D N T S D I S G RV5-G3 4 T T N N S W K D Q D A V D K Q D A N K D K D A T L S E A G RV5-G4 4 S T S T E W K D Q N L I D K Q D T A D T R A S G E S T S G LLR-G10 4 T T N N E W T S Q N A V D K Q N T G D T R N S S L S E A G 1 Circulating isolates belonged to lineage VI of evolutionary tree of VP7 gene; 2 Circulating isolates belonged to lineage III. 3 G9 genotype-specific vaccines. 4 non-G9 genotype-targeted vaccines. Numbers in red font, immune evasion-related neutralizing determinants. Letters with blue background, variations in neutralizing epitopes of global circulating isolates and vaccines relative to the 33 Chinese sample RVA strains in Lineage VI. The VP4 spike of RV is proteolytically cleaved into VP8* and VP5* subunits. The receptor-binding VP8* subunit harbouring four antigenic determinants (designated 8-1 to 8-4), while the membrane-penetration VP5* subunit containing five immunogenic clusters (5-1 to 5-5). Collectively, these domains encompass 37 critical residues related with neutralizing antibody recognition 31 . All dominant P[8]-genotype Wa-like isolates including the 38 Chinese strains and other G genotype strains all over the world, such as Moscow-40 (G3P[8]), Dhaka16 (G1P[8]), and CAU17L-103 (G8P[8]) exhibited nearly identical amino acid sequences across the eight epitope regions (8-1, 8-2, 8-3, 8-4, 5-1, 5-2, 5-3, 5-4, and 5-5). The only exceptions were a few strains with variations at positions 195, 113, and 135, with amino acids D/N/N, differing from the predominant sequences (with G/D/D). In comparison with the 38 Chinese G9P[8] RVA isolates, the P[8]-genotype vaccine RV1 exhibited six amino acid variations in the antigenic determinants 8-1/8-3 of VP4 at positions 150/195/113/125/131/135 (Table 5). Notably, the changes D150E and D135N might impact vaccine immunogenicity (Table 5). Similarly, the P[8]-RV5 vaccine displayed four aa variations compared to the 38 isolates in China, occurring at positions 150/195/113/388 in the VP4 antigenic determinants (Table 5). Among these, D150E and I388L were identified as potential escape mutations Table 5). For ROTAVIN-M1 rotavirus vaccine (P[8] genotype-targeted), three amino acid variations (150/195/113) were observed compared with the 38 Chinese G9P[8] RVAs (Table 5). The mutation D150E was also associated with escape mutant characteristics (Table 5). Additionally, a comparison of VP4 deduced determinants in heterologous P-genotype vaccines - including RV5, ROTAVAC and LLR (P[5]/P[11]/P[15]), revealed much more amino acid variations (19 to 22) relative to the 38 Chinese G9P[8] strains (Table 5). The structural mapping of the VP7 protein trimer revealed that the antigenic regions of 38 Chinese G9P[8] RVA strains from 2018 to 2020 each had four variant sites compared to both the G9 vaccines ROTASILL and ROTAVAC. In contrast, there were more pronounced and widely distributed 3D structural differences when compared to the vaccines RV1 (G1), Rotavin-M1 (G1), and LLR (G10) (Figure 6A). Analysis of the VP8* protein structure indicated that the number of variant sites between Chinese G9P[8] RVA strains and the P[8] vaccines Rotarix, RotaTeq, and Rotavin-M1 were 6, 3 (excluding I388L in the 5-1 region of VP5* subunit), and 3, respectively. These variants were non-uniformly distributed across the antigenic epitope regions 8-1 and 8-3, whereas regions 8-2 and 8-4 remained conserved (Figure 6B). Table 5 Presumed VP4 neutralizing antigen epitope differences between 38 Chinese Sample RVA strains and vaccine strains or P[8] strains circulating globally. 100 146 148 150 188 190 192 193 194 195 196 180 183 113 114 115 116 125 131 132 133 135 87 88 89 384 386 388 393 394 398 440 441 434 459 429 306 38 Chinese RVA strains D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R Z6398-CHN-G9P[8]-E2-2019 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R Fuzhou18-152-CHN-G9P[8]-2018 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R JS2016-CHN-G9P[8]-2016 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R Km15100-CHN-G9P[8]-2015 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R SC9-CHN-G9P[8]-2014 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R Tokyo18-43-JPN-G9P[8]-2018 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R VU12-13-101-USA-G9P[8]-2013 1 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R JES11-ITA-G9P[8]-2010 1 D S Q D S T N L N G I T A N P V D N R N D D N T N Y F I W P G R T P E L R CAU09-376-KOR-G9P[8]-2017 1 D S Q D S T N L N D I T A D P V D N R N D D N T N Y F I W P G R T P E L R Moscow-40-RUS-G3P[8]-2020 2 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R Dhaka16-BGW-G1P[8]-2003 2 D S Q D S T N L N G I T A D P V D N R N D D N T N Y F I W P G R T P E L R CAU17L-103-KOR-G8P[8]-2009 2 D S Q D S T N L N G I T A D P V D N R N D N N T N Y F I W P G R T P E L R RV1-G1P[8] 3 D S Q E S T N L N N I T A N P V D S S N D N N T N Y F I W P G R T P E L R RV5-P[8] 3 D S Q E S T N L N D I T A N P V D N R N D D N T N Y F L W P G R T P E L R Rotavin-M1-P[8] 3 D S Q E S T N L N D I T A S P V D N R N D D N T N Y F I W P G R T P E L R LLR-P[15] 4 D T N T Y T N Y D S V T A P E T T T A N P Q T S E Y F L W P G R T P D L R Rotavac-116E-P[11] 4 T S A A G Y N V P N A D G A Q T S T D N S S S N D Y F L W P G G T P Q C R RV5-P[5] 4 G A D D Y V N Y A S V T A T S E T S S N A D T G P Y F L W P G R T P E L R 1 Circulating G9P[8] isolates belonged to lineage 3 of VP4 ML tree. 2 Other circulating isolates belonged to lineage 3 with non-G9 genotype. 3 P[8] genotype-specific vaccine strains. 4 Non-P[8] genotype-targeted vaccines. Numbers in red font, amino acid sites linked to antigen escape. Letters with blue background, antigenic variations in reference strains compared to the 38 analyzed strains. For P[8] strains of other G genotypes in lineage 3, only a subset served as references. 4. Discussion Data analysis showed that between 2018 and 2020, RVA infection was a significant pathogen among hospitalized children under five years old with acute gastroenteritis in China (23.62%). RVA prevalence exhibited a distinct seasonal pattern, characterized by higher incidence in autumn and winter. G9P[8], as the predominant circulating G/P genotype combination, constituted 69.51% of all RVA cases. Additionally, the proportion of the G9P[8] genotype among RVA cases showed monthly variation, which may indicate differences in adaptability to seasonal environments among different RVA genotypes. The G9P [8] genotype combination is one of the major Group A rotaviruses transmitted worldwide 32 . G9P[8] were found in about 70% of paediatric patients admitted to hospitals for acute gastroenteritis by 2016 33,34 . Prior to 2016, G9P[8] strains generally exhibited a Wa-like genomic profile: I1-R1-C1-M1-A1-N1-T1-E1-H1. In this study, genome sequencing revealed that 38 randomly selected RVA samples from the China Rotavirus Surveillance Network across 20 provinces were identified as the G9P[8] genotype, with a third of the samples (13; 36.8%) belonging to the recently recognized new genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2). This new variant was initially observed in Japan in 2019, where it became the dominant strain locally 24 and quickly spread throughout Tokyo. Internal surveillance data from the China CDC Rotavirus Surveillance Network identified G9P[8]-E2 has been the primary RVA genotype combination in China since 2018. There was considerable homology among the 38 strains across the 11 gene segments in nucleotide and amino acid. Overall, these strains showed the greatest homology and the most intimate genetic connection when compared to the G9P[8] isolates from China and Japan in 2018 and 2019. The phylogenetic tree shows that the NSP4 gene of the 38 strains is distributed between two genotypes (E1 and E2), the VP7 gene is distributed across two G9 lineages (Lineage III and Lineage VI), and the distribution of other genes is relatively concentrated, except for one lineage 4 strain in VP4). These results suggest that the G9P[8] RVA strains circulating in China exhibit a low level of genetic diversity and relative genetic stability. The 13 Chinese G9P[8]-E2 isolates showed the highest nt/aa identities with Japanese G9P[8]-E2 isolates. Their NSP4 genes also had closest homology to Japan’s classic DS-1 G2P[4] isolates. These suggest Chinese and Japanese G9P[8]-E2 RVAs might share a common origin, likely from reassortment between Japanese G9P[8] and G2P[4] rotaviruses, consistent with previous finding 20 . Studies show Wa-like/DS-1-like RVs dominate in ~10-year cycles 35 . Host adaptation to one genomic constellation may drive immune resistance, while another evolves to enhance transmissibility. The presence of G9P[8]-E2 in Chinese RVA suggests a transition from Wa-like to DS-1-like strains. Co-circulation of both constellations may increase reassortment opportunities, fostering new strains. Thus, G9P[8]-E2’s dominance may not be permanent, though ongoing surveillance is needed to confirm trends. Additionally, studies suggest RV genotype infection preferences may link to host histo-blood group antigens (HBGA), potentially causing dominant RV genotype variations across populations/countries 14,15,36 . Even within the same genotype, viral strains show significant genetic variations by ethnicity/region 24,37 . Collectively, G9P[8]-E2 may not globally dominate from a geographic perspective. Before G9P[8]-E2 emerged, Japan reported G3P[8]-E2 reassortants at high prevalence (28% and 28.6% of RV genotypes, respectively) 24 . Such high proportions of DS-1/Wa-like reassortments are rare for other genotypes/fragments, suggesting these strains gained strong environmental and host adaptability recently. The E2-encoded NSP4 protein acts as an enterotoxin and aids rotavirus replication via VP5*-mediated DLP formation 38 . Its crucial biological role during RV infection might explain why DS-1-like NSP4/E2 readily reassorts with Wa-like genomes. Based on this, we hypothesize that novel reassortant with other Chinese strains such as G1P[8]-E2 and G12P[8]-E2 may emerge in the future, warranting ongoing surveillance of their spread and epidemic potential. With global vaccine use potentially altering viral prevalence and evolution rates, accurately estimating G9P[8] RVA evolution is key to understanding genotype dynamics. This study found G9’s evolution rate at1.402 × 10 -3 substitutions/site/year, matching prior research 32,39 . G9’s VP7 evolved faster than G1-G3 (5.99–8.63 × 10⁻⁴/5.66–9.196 × 10⁻⁴/7.34 × 10⁻⁴ substitutions/site/year) 28 . The P[8] rate (6.924×10⁻⁴ substitutions/site/year) aligned with G1P[8] estimates but lagged behind G9 40 . Rotavirus genotype shifts and evolution rate changes might be linked to vaccine introduction in various regions 35,41,42 . Global vaccines like Rotarix™ and RotaTeq™, plus China’s LLR vaccine, do not target G9. While RVA’s antigenic diversity may evade immunity 43 , no clear evidence shows vaccines directly drive G9’s prevalence via increased evolution or selective pressure. Additionally, facing selective pressures and evolutionary rate changes, rotavirus may see global spread of new genotypes/sub-lineages. This study found significant differences between the China/Japan G9 lineage (lineage IV) and the global G9 lineage (lineage III), consistent with previous study 44 , needing further investigation. Similar selective pressures drive convergent evolution, often leading to variant epitope mutations (especially in neutralizing sites) that evade immunity 18,19 , underscoring the need for antigen-matched vaccines 43 . This study compared VP7 neutralizing epitopes of 38 Chinese G9P[8] RVA strains to vaccines. Only two amino acid differences (N100D, S221G) were found among 38 strains, indicating high antigenic conservation in 2018–2020 circulating strains. G9-homologous vaccines showed many VP7 epitope differences (ROTASIIL: 87, 100, 221, 242; ROTAVAC: 87, 100, 145, 221), with mutations at 87, 100, and 145 potentially enabling immune escape. P[8]-homologous vaccines also had more VP4 epitope variations compared with Chinese strains (RV1: 150, 195, 113, 125, 131, 135; RV5: 150, 195, 113, 388; ROTAVIN-M1: 150, 195, 113), with 150, 135, and 388 impacting immunogenicity. Larger epitope gaps were seen with non-G9/P[8] vaccines. However, studies indicated such differences have not been shown to significantly diminish vaccine efficacy 45 . Nevertheless, ongoing clinical monitoring of vaccine effectiveness is critical given these variations. In summary, RVA was a significant pathogen in hospitalized Chinese under-5 children with acute gastroenteritis, showing distinct seasonality with autumn and winter peaks. Epidemiological analyses and random sampling showed G9P[8] is China’s dominant rotavirus strain, with G9P[8]-E2 emerging as a key lineage. VP4/VP7 homology, phylogenetic, Bayesian evolutionary dynamics analysis, and neutralizing epitope studies revealed significant genetic and antigenic divergence between current vaccines and circulating G9P[8] strains. Therefore, on the one hand, it is crucial to continuously conduct clinical monitoring of vaccine effectiveness, and on the other hand, developing new vaccines based on China’s G9P[8] RVA strains may better address local immunization needs. 5. Conclusion Data analysis showed that between 2018 and 2020, RVA infection was a significant pathogen in Chinese hospitalized children under five with acute gastroenteritis, with distinct seasonal prevalence favoring autumn and winter. Epidemiological analysis and random sampling show that G9P[8] is the predominant rotavirus genotype in China, with G9P[8]-E2 emerging as a key lineage. Phylogenetic analysis indicates that China’s G9P[8]-E2 rotavirus most likely originated from a genetic reassortment event between Japanese G9P[8] and G2P[4] rotaviruses; however, its dominance may not be permanent, and it is unlikely to become globally predominant. VP4/VP7 homology, phylogenetic, Bayesian evolutionary dynamics analysis, and neutralizing epitope studies revealed significant genetic and antigenic divergence between current vaccines and circulating G9P[8] strains, underscoring the critical need for continuous clinical monitoring of vaccine efficacy, while developing new vaccines based on China’s G9P[8] rotavirus strains may better address local immunization needs. Abbreviations RVA: Group A rotavirus; RVGE: Rotavirus gastroenteritis; nt: nucleotide; aa: amino acid Availability of data and materials All sequences used in this study are available in GenBank. All other relevant information is provided in this current manuscript. If required, the data presented in this work can be shared by e-mail. Ethics approval and consent to participate This study was reviewed and approved by the National Institute for Viral Diseases Control and Prevention (Beijing, China). The guardians of the recruited children in this study were informed of the aims of this investigation and provided oral consent. Authors’ contributions Z.-J.D. and D.-D.L. conceived and designed the experiments; R.P. and M.-X.W. performed the experiments; R.P. and J.-B.X. analysed the data; R.-R.C., C.-X.L., X.L., X.-Z.K., Y.-H.C., M.-R.T., Z.-Y.F., Y.-G.Z., X.H., W.-N.Z., and P.W. collected samples; R.P. wrote the main text; All authors reviewed the manuscript. Funding National Key Research and Development Program of China. Development, Safety and Efficacy Evaluation of Oral Vaccines for Respiratory Diseases (Finance Code 2022YFC2304302); National Key Research and Development Program of China. Development and Production Technologies for Vaccines against Newly Emerging and Sudden Severe Infectious Diseases for Human Use (Finance Code 2018YFC1200602). Acknowledgements The authors thank the contributions by the staff at the National Institute for Viral Diseases Control and Prevention, Chinese Centre for Disease Control and Prevention. Competing interests The authors declare that they have no conflict of interest. Author details 1 National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Viral Disease Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing 102206, China. [email protected] (R.P.); [email protected] (M.X.W.); [email protected] (D.-D.L.); [email protected] (Z.-J.D.) 2 Department of Poliomyelitis, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. 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Genomic and evolutionary characteristics of G9P[8], the dominant group a rotavirus in China (2016–2018). Front Microbiol . 2022;13(September). doi:10.3389/fmicb.2022.997957 45. Kulkarni R, Arora R, Arora R, Chitambar SD. Sequence analysis of VP7 and VP4 genes of G1P[8] rotaviruses circulating among diarrhoeic children in Pune, India: A comparison with Rotarix and RotaTeq vaccine strains. Vaccine . 2014;32(S1):A75-A83. doi:10.1016/j.vaccine.2014.03.080 Figure Legends Figure 1 The 38 RVA strains collected from sentinel hospitals in 12 provinces and municipalities in China from 2018 to 2020. Figure 2 Monthly prevalence of China’s RVA from 2018 to 2020. (A) The positive ratio of RVA by ELISA among all diarrhoea from under 5 years old. (B) The positive ratio of G9P[8] genotype among all diarrhoea from under 5 years old. (C) The composition ratio of G9P[8] genotype in the positive RVA. Figure 3 Phylogenetic analysis of the VP7, VP4, VP6 and NSP4 segments from the study strains. (A) VP7 tree. Genotype G2 (RotaTeq vaccine and DS-1 strains) were used as outgroup for VP7-G9 tree. (B) VP4 tree. Genotype P[8] (Rotavac-116E strain) was used as the outgroup for VP4-P[8] tree. (C) VP6 tree. (D) NSP4 tree. The trees were made using maximum likelihood with 1000 number of bootstraps. Numbers at the nodes are no less than 70. Figure 4 Phylogenetic analysis of the VP1-VP3, NSP1-NSP3 and NSP5 segments from the study strains. (A) VP1 tree. (B) VP2 tree. (C) VP3 tree. (D) NSP1 tree. (E) NSP2 tree. (F) NSP3 tree. (G) NSP5 tree. The trees were made using maximum likelihood with 1000 number of bootstraps. Numbers at the nodes are no less than 70. Figure 5 Root-to-tip divergence analysis and MCC topologies for G9P[8] VP7 and VP4 inferred by Bayesian evolutionary analysis employing MCMC chains. (A) Root-to-tip divergence analysis of VP7. (B) Root-to-tip divergence analysis of VP4. (C) VP7 MCC tree. Contemporary Chinese variants were highlighted in blue. (D) VP4 MCC tree. Contemporary Chinese variants were highlighted in purple. The MCC trees integrated temporally annotated strains, including GenBank-derived references. Figure 6. Presumed surface-exposed antigenic epitope amino acid variations in VP7 trimers/VP8* proteins between Chinese G9P[8] RVA and vaccines. (A) VP7 trimer: 7-1a (red), 7-1b (purple), and 7-2 (green) are surface-exposed antigenic epitopes. The figure shows the amino acid variations (blue) in VP7 surface-exposed antigenic epitopes between Chinese G9P[8] RVA and G9-type vaccines (Rotasill, Rotavac) as well as G1-type vaccines (RV1 and ROTAVIN-M1), respectively. Due to the numerous differences from RV5, they are not shown here. (B) VP8* subunit: The upper and lower parts on the left show the front and back sides of the VP8* domain, respectively, with surface-exposed antigenic epitopes 8-1 (red), 8-2 (orange), 8-3 (purple), and 8-4 (green). The figure shows the amino acid variations (blue) in VP8* surface-exposed antigenic epitopes between Chinese G9P[8] RVA and P[8]-type vaccines (RV1, RV5, and ROTAVIN-M1), respectively. Table Legends Table 4 Predicted VP7 neutralizing antigen epitope polymorphisms of 38 RVA isolates in China compared to vaccines and global G9 circulating reference isolates. Table 5 Presumed VP4 neutralizing antigen epitope differences between 38 Chinese Sample RVA strains and vaccine strains or P[8] strains circulating globally. Supplementary Material File (table 4.docx) Download 44.60 KB File (table 5.docx) Download 39.72 KB Information & Authors Information Version history V1 Version 1 13 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords epidemiology evolution human rotavirus virus classification Authors Affiliations Rui Peng National Institute for Viral Disease Control and Prevention View all articles by this author Mengxuan Wang National Institute for Viral Disease Control and Prevention View all articles by this author Jinbo Xiao National Institute for Viral Disease Control and Prevention View all articles by this author Ranran Cao 0000-0001-5809-4714 National Institute for Viral Disease Control and Prevention View all articles by this author Caixia Li National Institute for Viral Disease Control and Prevention View all articles by this author Xiang Li National Institute for Viral Disease Control and Prevention View all articles by this author Xiaozhou Kuang National Institute for Viral Disease Control and Prevention View all articles by this author Yihui Cao National Institute for Viral Disease Control and Prevention View all articles by this author Meirong Tang National Institute for Viral Disease Control and Prevention View all articles by this author Zhongyan Fu National Institute for Viral Disease Control and Prevention View all articles by this author Yugeng Zhang National Institute for Viral Disease Control and Prevention View all articles by this author Xiao Hu National Institute for Viral Disease Control and Prevention View all articles by this author Wenna Zhao National Institute for Viral Disease Control and Prevention View all articles by this author Peng Wang National Institute for Viral Disease Control and Prevention View all articles by this author Dandi Li [email protected] National Institute for Viral Disease Control and Prevention View all articles by this author Zhaojun Duan National Institute for Viral Disease Control and Prevention View all articles by this author Metrics & Citations Metrics Article Usage 205 views 116 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Rui Peng, Mengxuan Wang, Jinbo Xiao, et al. 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